1. Field of the Invention
This invention relates to regulation of cell growth and proliferation and specifically to a novel cell cycle-related polynucleotide, 5'ALT, and novel polynucleotides encoding truncated cell cyclin inhibitors, p16.sup.INK4A and p15.sup.INK4B. The invention also relates to the identification of neoplastic cells in a sample, by detecting p16 mRNA, polypeptide, and 5'CpG island methylation of p16DNA.
2. Description of Related Art
The growth cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases ("CDKs"). The cyclins and their associated CDKs move cells through the three phases of the growth cycle (G1, S and G2, respectively) leading to division in the mitosis phase (M). The cyclin/CDK complexes whose role in cellular proliferation has been most clearly defined to date are the cyclin D/CDK enzymes, which are believed to assist in the progression of the G1 growth cycle phase. Of these enzymes, cyclin D1 is believed to be an oncogene, whose overexpression stimulates excessive cell division through the continuous production of kinase, thus contributing to the development of cancers of, for example, the breast and esophagus. Cyclin D1 is specifically bound by CDK4 as part of a multi-protein complex that also consists of a protein known as p21 and cell nuclear antigen. Known inhibitors of such cyclin/CDK overexpression include the tumor suppressor protein p53 and the protein product of the retinoblastoma (Rb) gene. Recently, two putative inhibitors of cell cyclins, p16.sup.INK 4A and p16.sup.INK4B, were isolated (Serrano, et al., Nature, 366:704, 1993; Hannon, et al., Nature, 371:257, 1994, respectively). The cyclin-CDK inhibitors p16.sup.INK4A (CDKN2MTS-1) and p15.sup.INK4B (MTS-2) are inportant components of cell cycle regulation. Transition through G1 is promoted by the cyclin-dependent protein kinases CDK4 and CDK6 which phosphorylate Rb resulting release of E2F and cell cycle progression (Hunter, T. & Pines, J.,Cell 79: 573-582, 1994). In addition to more universal inhibitors (Morgan, D. O., Nature, 374:131-134, 1995), these kinases are strongly inhibited both p16.sup.INK4A and p15.sup.INK4B. Isolation of the genes for these negative cell cycle regulators was quickly followed by their co-localization to chromosome 9p21, within a critical region commonly deleted in many types of human cancer (Kamb, A., et al., Science, 264:436-440, 1994; Nobori, T., et al., Nature, 368:753-756, 1994). Familial and sporadic malignant melanomas have been consistently associated with cytogenetic abnormalities of chromosome 9p21 (Fountain, et al., Proc. Natl. Acad. Sci., USA, 89:10557, 1992; Cannon-Albright, et al., Science, 258:1148, 1992) Deletions of this region are also common in gliomas (Olopade, et al., Cancer Res., 52:2523, 1992), lung cancers (Olopade, et al., Cancer Res., 53:2410, 1993), and leukemias (Olopade, et al., Genomics, 14:437, 1992). Although excellent tumor suppressor gene candidates, somatic point mutations were found to be rare in many primary human tumors with hemizygous loss of 9p21 (Cairns, et al., Science 245:415-416, 1994).
Frequent loss of heterozygosity (LOH) and homozygous deletion of chromosome 9p21 has suggested the presence of tumor suppressor genes in this region. Localization of an inhibitor of the cyckin D/cyclin dependent kinase 4 complex, now called CDKN2/p16, to 9p21, along with frequent homozygous deletions of this gene in human cancer cell lines, suggested that p16 might be the target gene (Kamb, et al, supra; Nobori, et al., supra). Since the initial reports of this homozygous deletion, numerous studies have shown varying, but in general much less frequent, abnormalities of p16 in primary tumors of these cancers. For example, although the rate of homozygous deletions ranged from 40-60% of breast cancer cell lines (Kamb, et al., supra; Xu, et al., Cancer Res., 54:5262, 1994), neither homozygous deletion or point mutations are typically observed in primary breast carcinomas. Also, certain common neoplasms, such as prostate and colon cancer, have not been found to harbor homozygous deletions in established cell lines.
In this regard, allelic loss of 9p21 has been found to occur early in the progression of at least two tumor types (Cairns, et al., Oncogene, 8:1083, 1993; van der Riet, et al., Cancer Res., 54:1156, 1994). Hemi- and homozygous losses of chromosome 9p21 are among the most common changes found in most tumor cell lines (Olopade, et al., Genomics, 14:437, 1992) and primary tumors (Merlo, et al. Cancer Res., 54:640, 1994; Migeon, et al., Genet Res., 56:91, 1990).
In eukaryotic cells, methylation of cytosine residues that are immediately 5' to a guanosine, occurs predominantly in CG poor regions (Bird, A., Nature, 321:209, 1986). In contrast, discrete regions of CG dinucleotides called CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation (Migeon, et al., supra) and parental specific imprinting (Li, et al., Nature, 366:362, 1993) where methylation of 5' regulatory regions can lead to transcriptional repression. De novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas (Sakai, et al., Am. J Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Latif, et al., Cancer Res., 52:1451, 1992). Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated 5'CpG island (Issa, et al., Nature Genet., 7:536, 1994, Herman, et al., Proc. Natl. Acad Sci., U.S.A., 91:9700, 1994).
Identification of the earliest genetic changes in tumorigenesis is a major focus in molecular cancer research. Diagnostic approaches based on identification of these changes are likely to allow implementation of early detection strategies and novel therapeutic approaches targeting these early changes might lead to more effective cancer treatment.